Abstract
Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.
Highlights
Platelets are anucleate cells that are important for haemostasis, thrombosis, and atherosclerotic disease
Previous proteomic studies of intact platelets have collectively identified hundreds of proteins using a variety of fractionation strategies including 2-dimensional electrophoresis (2DE), multidimensional chromatographic separations, membrane prefractionation techniques, and adsorption to combinatorial hexapeptide ligand libraries [1,2,3,4,5,6]
Platelet suspensions were supplemented with 1.8 mM CaCl2, incubated at 37◦C in an aggregometer under constant stirring conditions (1100 rev/min), and stimulated with 5 μM thrombin receptor-activating peptide (TRAP) for 3 minutes
Summary
Platelets are anucleate cells that are important for haemostasis, thrombosis, and atherosclerotic disease. Previous proteomic studies of intact platelets have collectively identified hundreds of proteins using a variety of fractionation strategies including 2-dimensional electrophoresis (2DE), multidimensional chromatographic separations, membrane prefractionation techniques, and adsorption to combinatorial hexapeptide ligand libraries [1,2,3,4,5,6]. A previous study from our laboratory using a MuDPIT (multidimensional protein identification technology) approach identified over 300 proteins secreted by platelets upon thrombin activation [1]. These proteins may modulate the interaction of platelets with their local cellular environment. Platelet releasate has previously been shown to induce endothelial cell permeability, endothelial cell chemotaxis, and corneal epithelial cell proliferation in cellular assays [7,8,9]
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